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Enamel matrix derivative induces connective tissue growth factor expression in human osteoblastic cells.

BACKGROUND: Enamel matrix derivative (EMD) stimulates the production of transforming growth factor-beta (TGF-beta), which has been suggested to play a role in mediating the effects of EMD in periodontal tissue regeneration. Connective tissue growth factor (CTGF) is a mediator of TGF-beta and promotes cell development. The interaction between EMD and CTGF is unknown. This study explored the effects of EMD on CTGF expression in human osteoblastic cells and whether the interaction is modulated by the TGF-beta signaling pathway. Also, the roles of CTGF in cell proliferation, cell cycle progression, and mineralized nodule formation of EMD-induced osteoblastic cultures were examined.

METHODS: Human osteoblastic cells (Saos-2) were treated with 25 to 100 microg/ml EMD with or without the addition of TGF-beta inhibitor. CTGF mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and CTGF protein levels were assayed by Western blot analysis. In addition, cell cycle progression and DNA synthesis were determined by flow cytometry and 5-bromo-2'-deoxyuridine (BrdU) incorporation following EMD treatment with or without CTGF antibody. Mineralization was examined by alizarin red staining and quantified by elution with cetylpyridinium chloride.

RESULTS: Western blot and RT-PCR analysis demonstrated a dose-dependent increase of CTGF expression by EMD. EMD-induced CTGF expression was reduced significantly in the presence of TGF-beta inhibitor. Cell cycle and BrdU analysis revealed an increase in cell proliferation following EMD treatment, which was due to an increase in the percentage of cells in the G2/M phase of the cell cycle. No significant effect was found when anti-CTGF antibody was added. Conversely, mineralization was inhibited significantly in EMD-treated cultures in the presence of anti-CTGF antibody.

CONCLUSIONS: EMD stimulates CTGF expression, and the interaction is modulated via TGF-beta in osteoblastic cells. Also, CTGF affects EMD-induced osteoblastic mineralization but not cell proliferation. To our knowledge, these results provide novel insight into EMD-CTGF interaction, two biomodifiers that have therapeutic relevance to tissue engineering and regeneration.

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